The study of protein interaction networks is an important approach to understanding cell biology and represents a growing interest in drug discovery. However, developing sensitive, quantitative, and high-throughput screening (HTS) methods that permit direct measurements of protein-protein interactions has proven challenging. The traditional co-immunoprecipitation approach is not practical for HTS as it requires time-consuming sample processing and detection, including Western blotting.

We have developed the Thermo Scientific™ Pierce™ Quantitative Immunoprecipitation (qIP) System to measure interactions between pairs of recombinant proteins expressed in mammalian cells. The highly sensitive luciferase assay method eliminates the need for gel electrophoresis and Western blotting; furthermore, the technique is amenable to HTS. Here we briefly describe the Pierce qIP Assay System and then demonstrate its utility by presenting results from several example experiments, including one performed as an HTS compatible assay in 384-well plates which yielded high signal-to-noise ratios and Z’ values.

Results and discussion

The Pierce qIP assay depends upon a small, bright luciferase, Thermo Scientific™ TurboLuc™ (Tluc) Luciferase, which is fused to a protein of interest and transiently co-expressed with an epitope-tagged (e.g., HA or Myc tag) protein in mammalian cells. Cells expressing both proteins are lysed, and the interaction (HA-protein X with Tluc-protein Y) is captured onto a solid support, typically either anti-tag agarose or magnetic beads. (In this study, we also demonstrate the method using anti-tag coated microplate wells.) The protein interaction (between X and Y proteins) is quantified by measuring the Tluc luciferase activity of the pull-down product.

To test the interaction of two proteins of interest (X and Y) in this assay system, genes for X and Y must be cloned into two separate expression vectors; gene X into an epitope-tagged vector (HA- or Myc-tag), and gene Y into Tluc-fusion vector. Upon co-transfection into mammalian cells, the vectors express the X and Y proteins, which then interact based on treatment conditions. Next, the cells are lysed, and the target protein-complex is immunoprecipitated using anti-epitope-tag beads. Finally, the co-IP product (protein Y) is quantified by measuring Tluc luciferase activity (Figure 1).

Figure 1. The Pierce qIP assay overview. Schematic of qIP assay mechanism and resulting data. This example uses HA as the epitope tag and anti-HA agarose resin. In the test condition (left), Tluc-tagged protein Y co-immunoprecipitates via its interaction with HA-tagged protein X. The resulting luminescence signal is directly proportional to the amount of protein Y bound to protein X (i.e., the abundance of the X-Y protein interaction). In the negative control (right), Tluc-tagged protein Y does not interact with HA-tagged RFP (red fluorescent protein) and is not pulled down, resulting in detection of only the background luminescence signal (noise). The protein interaction is represented by the normalized signal-to-noise ratio. RLU = relative luminescence units.

In addition to the standard workflow using bead-based co-IP (Figure 2A), we also developed a plate-based co-IP procedure that amenable to HTS (Figure 2B).

A. Bead-based qIP assays

B. Plate-based qIP assays

Figure 2. Pierce qIP assay workflows. The co-immunoprecipitation (capture) step of the qIP assay procedure can be performed using agarose or magnetic beads (A) or coated microplates (B).

The Pierce quantitative immunoprecipitation (qIP) system depends on TurboLuc luciferase enzyme (Tluc) to accurately and precisely reflect the abundance of a specific co-IP product without time-consuming Western blot and band densitometry steps. Tluc luciferase is a small (18kDa) intracellular luciferase expressed by a synthetic gene designed based on the luciferase from Metridinidae copepods. Tluc luciferase uses coelenterazine as substrate and has been engineered to provide bioluminescence that is much brighter than commonly used firefly and Renilla luciferases. The intense bioluminescence greatly enhances sensitivity of Tluc luciferase-based assays, enabling detection of very minute amounts of luciferase (signal) in qIP assays. In addition, the Tluc luciferase gene is codon optimized for mammalian cell expression.

To demonstrate the excellent sensitivity of Tluc luciferase as a reporter for the qIP protein interaction assay, we benchmarked Tluc against three conventional luciferases, WT Renilla, Green Renilla, and intracellular Gaussia (icGaussia) in the qIP assay system (Figure 3). We cloned two genes (p53 and Bcl-xL) into equivalent expression vectors with each luciferase. Then, we performed qIP assays using each luciferase vector with the respective HA-tagged interactors (p53 and BAD) of p53 and Bcl-xL. Tluc activity in qIP assay with p53-p53 pair was 2.3 million RLU, which is 15 times greater than the intracellular Gaussia (0.15 million RLU). Tluc activity in qIP assay with BAD/Bcl-xL pair was 15.6 million RLU, which is 5 times greater than the intracellular Gaussia (3.19 million RLU).

Figure 3. Tluc luciferase as the basis of the Pierce qIP assay. Tluc luciferase was benchmarked against three conventional luciferases, WT Renilla, Green Renilla, and intracellular Gaussia (icGaussia) in qIP assays (anti-HA agarose bead method). Blue (left Y-axis) represents the p53/p53 protein interaction pair as model system. Tluc activity in qIP assay with p53-p53 pair was 2.3 million RLU, which is 15 times greater than the intracellular Gaussia (0.15 million RLU). Green (right Y-axis) represents the BAD/Bcl-xL protein interaction pair as model system. Tluc activity in qIP assay with BAD/Bcl-xL pair was 15.6 million RLU, which is 5 times greater than the intracellular Gaussia (3.19 million RLU).

To demonstrate the wide dynamic range of the Pierce qIP assay, we measured three very different known protein-protein interaction pairs (IRF3/HPV16 E6, p53/p53, and BAD/Bcl-xL) with the system (Figure 4). The RLU value ranged from 5,000 to 15 million (5-orders of magnitude), showing an excellent dynamic range to quantify protein-protein interaction pairs.

Figure 4. Ability to detect a wide range of protein-protein interaction pairs. Three known protein-protein interaction pairs (IRF3/HPV16 E6, p53/p53, and BAD/Bcl-xL) were expressed and quantified in the qIP assay. The RLU values ranged from 87 thousand to 15 million (180-fold; 2.25 orders of magnitude), showing an excellent dynamic range to quantify protein-protein interaction pairs (actual RLU values shown on the top of the bar graph).

A simple experiment with SMAD2 and SMAD4 demonstrates the ability of the Pierce qIP assay to accurately measure protein interactions that are dependent upon known treatment conditions affecting post-translational modification (Figure 5).

Figure 5. The Pierce qIP assay detects post-translational modification (PTM)-dependent protein-protein interactions. Left. Upon TGFβ binding to its receptor on the membrane surface, SMAD2 is phosphorylated by the activated receptor kinases and the phosphorylated form of SMAD2 forms a heterocomplex with the SMAD4. The SMAD2/SMAD4 complex is then translocated into the nucleus, where it binds transcription factors and activates transcription of TGFβ-response element dependent genes. Right. The PTM-dependent protein interaction between SMAD2 and SMAD4 increases approx. 5-fold after TGFβ1 treatment in HEK293T cells (3 hours after the ligand treatment).

Finally, we demonstrate that Pierce qIP assays can be performed using plate-based IP, i.e., capturing the expressed protein interaction directly to wells of anti-tag antibody-coated 96-well or 384-well plate instead of agarose or magnetic beads (Figure 6). We used our positive (BAD/Bcl-xL) and negative (RFP/Bcl-xL) controls to test the plate-based implementation of the Pierce qIP assay. The normalized QIR value (approx. 300) for BAD/Bcl-xL from the microplate-based assay is close to the value (approx. 500) from the resin-based assay, indicating that the sensitivity of both methods is compatible although the absolute RLU values are different due to the reaction volume.

Figure 6. 384-well microplate qIP protocol results in a robust assay amenable to HTS. Cells were grown in 6-well culture dishes at a concentration of 1.6 million cells per 2mL media. Plasmids (1.5μg each) were transfected and incubated for 24 hours. After cell lysis, cleared cell lysates were incubated in 384-well microplates (6 separate plates, 15 wells each) coated with anti-HA antibody at a concentration of 5μg/mL. The Tluc activity measurement was performed as described in the Methods section.

The quantitative immunoprecipitation (qIP) system depends on Tluc luciferase enzyme to accurately and precisely reflect the abundance of a specific co-IP product without time-consuming gel electrophoresis, Western blot and band densitometry steps.

The signal output (RLU) of the qIP protein interaction assay has a wide dynamic range (180-fold; 2.25 orders of magnitude), enabling detection of a wide range of affinities of protein-protein interaction pairs.

The microplate-based qIP assay, in which the pull-down (capture) step of the co-IP is performed directly in coated 384-well plates, results in a robust assay that is amenable for HTS.

Methods

Bead-based qIP assay

Assay reagents used in this procedure are identical to those supplied in the Pierce Agarose qIP Protein Interaction Kit, Tluc and HA Tags (Part No. 82032). 293T cells were plated at a concentration of 1.6 million cells/2mL into each well of a 6-well plate. DNA constructs (1.5μg each) were transfected to each well in a total volume of 200μL serum free media. After 24 hours, cells were lysed with vortexing and the clear lysates were added to microspin columns for pull-down. After approx. 3 hours, the resins were washed with the qIP assay reagents and resuspended in 160µL (see Figure 2). Finally, 20µL of the resuspended resins were transferred to wells of a black 96-well microplate, and the bioluminescence was measured after adding 60μL of prepared Tluc luciferase assay reagent (Part No. 82015).

Microplate-based qIP assay

293T cells, 1.6 x 10^6, were plated in 6-well culture plate and incubated overnight at 37°C. Transfection reaction mixture was prepared with 1.5μg of each DNA construct, HA-tagged BAD and Tluc-tagged Bcl-xL, and 6μL of TurboFect Transfection Reagents in a total volume of 200μL serum free media. The transfection mixture was added to each well. The control transfection mixture was prepared with HA-tagged RFP and Tluc-tagged Bcl-xL. After 24 hours of post-transfection, the cells were harvested by trypsin treatment and lysed with 160μL of the qIP lysis buffer. The total lysates were diluted with 360μL of the dilution buffer (1:3 ratio of the qIP lysis buffer: dilution buffer).

A black 386-well microplate was coated overnight at 4°C with 5μg/mL anti-HA monoclonal antibody in carbonate/nicarbonate coating buffer. The microplate was incubated with cell lysate by rocking for 2 hours. Plates were then washed three times in Thermo Scientific Multidrop Combi (#5840320) with washing buffer, which is a 1:3 mixture of qIP assay lysis buffer (Part No. 82013) and dilution buffer (Part No. 82014). Finally, the bioluminescence (RLU) was measured after adding 60μL of Tluc luciferase assay reagent (Part No. 82015).